There has been considerable interest in catabolism of the essential branched chain amino acids (leucine, isoleucine, valine) because of the nutritional, biochemical and clinical significance of these metabolites. Body concentrations of branched chain amino acids are regulated as evidenced by the severe metabolic consequences of disorders that result in excess or disproportionate amounts of these amino acids. The first step in the catabolic pathway is transamination catalyzed by the branched chain aminotransferase to form the branched chain alpha-keto acids. Our laboratory has established that there are two transaminase isoenzymes- - mitochondrial (BCAT-m) and cytosolic (BCAT-c). As part of our goal of defining the physiological role of these proteins, we will conduct detailed kinetic analyses of the purified enzymes. To understand the link between branched chain amino acid transamination, mitochondrial transport and body nitrogen metabolism, an in vitro model of cellular metabolism will be developed using isolated mitochondria and, in the future, intact cells. After transamination, oxidation of the branched chain alpha-keto acids occurs in the mitochondria. Transport of these alpha-keto acids (and subsequent metabolites) must occur across the mitochondrial inner membrane. We have characterized a specific mitochondrial transport system for branched chain alpha-keto acids which is separate from the second monocarboxylate transporter, the pyruvate carrier. Kinetically, however, these transporters are very similar. Purification of the branched chain alpha-keto acid transporter has resulted in the novel discovery that the mitochondrial aminotransferase, BCAT-m, is a bifunctional protein catalyzing transport of branched chain alpha-keto acids and transamination. Our goal is to determine the mechanism of BCAT-m catalyzed transport and the molecular architecture of the active site. This study will include using covalent modifying reagents to identify amino acid residues that participate in the transport and transaminase activities of BCAT-m. Experiments will be initiated to determine the crystal structure of the branched chain aminotransferases isoenzymes. As part of our third goal of developing a molecular model of mitochondrial monocarboxylate transport, we will clone the cDNA for the pyruvate transporter. This study should provide new information on the diversity of anion transport mechanisms.